PROJECT SUMMARY The biological underpinnings through which changes in diet or exercise influence healthspan - increased lifespan in the absence of disease - are not fully understood. Recent data in flies and worms have highlighted a potential role for lipolysis, the catabolism of triacylglycerol (TAG) stored within lipid droplets (LDs), as a factor that promotes healthspan. However, the mechanisms linking lipolysis to alterations in healthspan are not known. In addition, no studies have evaluated the role of lipolysis in healthspan regulation in mammals or the interaction of lipolysis and diet, which largely determines the composition of LDs and, therefore, their signaling properties. Thus, the objective of this application is to define the mechanisms through which lipolysis influences healthspan and to determine the contribution of dietary lipid composition to these effects. We hypothesize that lipolysis is a key signaling node that drives healthspan through alterations in fatty acid signaling. We base this hypothesis on preliminary data from our laboratory defining a signaling axis linking adipose triglyceride lipase (ATGL)-catalyzed lipolysis to sirtuin 1 (SIRT1) activity and downstream signaling to activate forkhead box protein 01 (FOXO1). We also show that overexpression of the Drosophila homolog of ATGL, brummer (Bmm), increases lifespan consistent the known roles of SIRT1 and FOXO1 as key regulators of lifespan. To test our hypothesis, we will utilize the strengths of both Drosophila and mouse models to test the following specific aims: Aim 1: To delineate the signaling pathway through which lipolysis influences healthspan in Drosophila. We will use Drosophila models to dissect the linearity of the Bmm-Sir2 (fly SIRT1 homolog)-dFOXO (fly FOXO1 homolog) signaling axis in regulating healthspan. Aim 2: To determine the interactions between diet and ATGL on healthspan in mice. These studies will explore the synergy between dietary lipid composition and ATGL overexpression on lifespan and aging-related declines in metabolism in mice. Aim 3: To characterize the interaction between ATGL and autophagy. In this aim, will test the effects of ATGL on autophagy and the role of autophagy in mediating the signaling and lifespan extending effects of ATGL/Bmm in both fly and mammalian models. These studies will be the first to comprehensively define the role of ATGL-catalyzed lipolysis in healthspan and how it interacts other physiological factors involved in lifespan regulation. These studies are innovative because they will use novel fly and mouse models to link individual factors known to influence lifespan such as diet into a cohesive model that better explains healthspan regulation. The proposed work is significant because it will greatly advance our understanding into the understudied area of lipid metabolism as a factor contributing to aging and will provide substantial insights into new or improved dietary, behavorial or pharmaceutical avenues to promote healthspan.